Repair processes in the yeast Saccharomyces cerevisiae are under extensive genetic control involving over 50 genes; among these are genes that function in recombinational repair, as well as normal meiotic and mitotic recombination. (Kunz, B. A., and R. H. Haynes Annu. Rev. Genet. 15:57-89 (1981); Game, J. C. (1983) in: Yeast Genetics, Fundamental and Applied Aspects (eds. Spencer, J. F. T., Spencer, D., and Smith, A.), pgs. 109-137, Springer-Verlag New York, Inc., New York; and Resnick, M. A. in: Meiosis (Ed. Moens, P.), pgs. 157-212 (1987), Academic Press, New York). Deoxyribonucleases are expected to play a role in many repair processes since they enable the excision of damaged DNA and provide a means for heteroduplex formation and processing in recombination. Several deoxyribonucleases have been shown, both genetically and biochemically, to function in recombination and repair. For example, the nuclease activity associated with the Escherichia coli recBCD proteins is required for much of host recombination and also for the chi-stimulated lambda bacteriophage (Chaudhury, A. M., and G. R. Smith, Proc. Natl. Acad. Sci. (USA) 81:7850-7854 (1984)). Holloman and Holliday (J. Biol. Chem. 240:8107-8113, (1973)) have described nuclease .alpha. from the eukaryote Ustilago maydis that is required for recombination and DNA repair. Because of a complex genetic control mechanism, involving two genes, the specific role of this nuclease has not been ascertained.
An endo-exonuclease from Neurospora crassa has also been implicated in recombination and repair (Chow, T. Y.-K, and M. F. Fraser, Can. J. Biochem. 57:889-901 (1979); Chow, T. Y.-K, and M. F. Fraser, J. Biol. Chem. 258:12010-12018 (1983); and Ramotar, D., et al., J. Biol. Chem. 262:425-431 (1987)). The phenotypes of mutants deficient or altered in nuclease activity include meiotic sterility and sensitivity to ultraviolet light, X-rays and/or alkylating agents (Fraser, M. J., et al. in: DNA Repair and Mutagenesis in Eukaryotes (Generoso, et al., eds.), pgs. 63-74 (1990), Plenum Publishing Corp., New York). A similar endo-exonuclease has also been isolated from Aspergillus nidulans (Koa, H., et al., Biochem. Cell. Biol. 68:387-392 (1990)) and from mammalian mitochondria (Tomkinson, et al., Nucl. Acids Res. 14:9579-9593 (1986)).
An endo-exonuclease, RhoNUC, from S. cerevisiae was isolated, characterized, and suggested to function in both repair and recombination (Chow, T. Y.-K, M. A. Resnick, J. Biol. Chem. 262:17659-17667 (1987); and Chow, T. Y.-K, land M. A. Resnick, Molec. Gen. Genet. 211:41-48 (1988)). Several properties of RhoNUC resemble those of the E. coli recBCD nuclease. The RhoNUC activity in both mitotic and meiotic cells is greatly influenced by a functional RAD52 gene. The RAD52 gene product is required for mitotic and meitic recombination and for the repair of double stranded breaks in DNA. In rad52 mutants, the mitotic level of the endo-exonuclease is less than 10% of the wild type level, and no increase is observed during meiosis (Resnick, M. A., et al., Molec. Cell. Biol. 4:2811-2817 (1984)).
In the past decade, recBCD-like eukaryotic deoxyribonucleases have been discovered which act on both single-stranded DNA (endonucleolytic) and double-stranded DNA (exonucleolytic) and were thus termed endo-exonucleases. These peptides were mostly exonucleolytic with a small endonucleolytic activity.
The endo-exonucleases isolated from the various eukaryotic sources studied so far share common antigenic epitopes with the E. coli recBCD nuclease. Rabbit polyclonal antibody raised against the Neurospora crassa endo-exonuclease cross-reacts with the endo-exonucleases from other species (Fraser, M. J., Chow, T. Y.-K., Cohen, H. and Koa, H., Biochem. Cell. Biol., Vol. 64, pp. 106-116 (1986); and Fraser, M. J., Koa, H. and Chow, T. Y.-K, J. Bacteriol., Vol. 172, pp. 507-510 (1990)).
A new model has been proposed by Rosenberg and Hastings (Rosenberg, S. M. and Hastings, P. J., Biochimie, Vol. 73, pp. 385-397 (1991)) in which endo-exonuclease action is an integral part of the recombination process. Unfortunately, no one has yet been able to isolate mammalian endo-exonucleases. To prove this model it would be helpful to analyze endo-exonucleases from various species. The importance of each region of the enzyme can then be estimated depending on the amount of conservation detected in each species' peptide.